The goal of our studies of T lymphocytes is to identify novel and functionally important proteins (enzymes, membrane channels, receptors) and to use their specific inhibitors, agonists, and antagonists in the development of new immunomodulating strategies. Nontoxic immunosuppressive drugs are needed to treat autoimmune diseases and to prevent rejection of transplanted organs. Our "extra-cellular ATP" hypothesis led us to explore the possible use of analogs of physiologically abundant molecules of ATP and adenosine in modulating the immune response. Subsequently, we described differential effects of extra-cellular ATP and adenosine on different subsets of thymocytes and T cells. These studies led to considerations of extra-cellular adenosine and ATP in regulation of normal T-cell differentiation and effector functions. We found that extra-cellular adenosine was efficient in antagonizing TCR-induced processes in T cells, thereby displaying the property of an immunosuppressive agent. This led to testing the possibility of modulating T cell generation and functions using extra-cellular adenosine. Using normal and TCR-transgenic animal models as well as polyclonal ex vivo lymphocytes and in vitro-maintained cell lines and clones, we found that low concentrations of extra-cellular adenosine strongly inhibited the TCR-triggered proliferation and upregulation of IL-2 receptor chain (CD25) molecules. These effects of extra-cellular Ado are likely to be mediated by A2a receptor-mediated signaling, since: i)poorly metabolized adenosine analogs cause the accumulation of cAMP and strong inhibition of TCR-triggered CD25 upregulation; ii)the A2a, but not the A1 or A3, receptors are the major expressed and functionally coupled adenosine receptors in mouse peripheral Tlymphocytes, and the adenosine-induced cAMP accumulation in lymphocytes correlates with the expression of A2a receptors; iii) the specific agonist of A2a receptor, CGS21680, induces increases in [cAMP]i in lymphocytes, while the specific antagonist of A2a receptor, CSC, inhibits the effects of Ado and CGS21680; and iv)the increases in [cAMP]i mimic the adenosine-induced inhibition of TCR-triggered CD25 upregulation and splenocyte proliferation. The additional enhancement of immunosuppressive effects of adenosine is expected from the use of adenosine kinase inhibitors, which may increase concentration of endogenous adenosine and by simultaneous addition of deoxycoformycin, an ADA inhibitor that is currently in clinical use.